Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) are present in the extracellular matrix (ECM) as well as on the cell surface. They play an important role in regulating cellular processes such as cell adhesion, migration, differentiation and proliferation (1–3). In addition, they interact with many molecules including growth factors (e.g. fibroblast growth factors and platelet-derived growth factor), cytokines, extracellular matrix proteins, lipoproteins, and β-amyloid proteins (2, 4–8). Release of these proteins by proteases or glycosidases provides a regulatory mechanism for induction of growth, chemotaxis, and extravasation of cells in normal and disease processes (3, 5).
Heparanase is an endo-(-D)-glucuronidase that degrades heparan sulfate proteoglycans (HSPG) at specific sites. Its activity has been detected in a number of cell types including human platelets, fibroblasts, neutrophils, activated T-lymphocytes, monocytes and human umbilical vein endothelial cells (HUVEC) (9–12). In addition, elevated levels of heparanase are associated with melanoma and other types of tumor cells (13–16). Evidence suggests that cleavage of the HS chains from HSPG by heparanase leads to the disassembly of the ECM and facilitates cell migration. It is an important process in the tissue invasion by blood-borne malignant tumor cells and leucocytes (14, 17–19). In fact, treatment of experimental animals with heparanase inhibitors considerably reduced the incidence of lung metastases (20–22), indicating that heparanase inhibitors may be applied to inhibit tumor cell invasion and metastasis.
The ability of activated lymphocytes, macrophages and granulocytes to penetrate ECM and migrate to target tissues was found to be dependent on heparanase activity (23). In response to various activation signals (e.g., immune complexes, antigens, nitrogens), heparanase is released from intracellular compartments (e.g., lysosomes, specific granules), suggesting its involvement in inflammatory and autoimmune responses. Treatment of experimental animals with heparanase inhibitors markedly reduced the incidence of T-cell mediated delayed-type hypersensitivity, experimental autoimmune encephalomyelitis and adjuvant arthritis (23–24), indicating that heparanase inhibiting compounds may be applied to inhibit autoimmune and inflammatory diseases.
Thus, heparanase plays an important role in the degradation of the extracellular matrix. It is implicated in inflammation, tumor angiogenesis and metastasis. Despite evidence implicating the attractiveness of heparanase as a therapeutic target for intervention, drug development has been hampered by the lack of a high throughput method for testing the ability of agents to inhibit heparanase activity.
Heparanase assays have been performed using radiolabeled ECM-associated HSPG (34–35). Radiolabeled material released from the ECM by heparanase cleavage is detected. Disadvantages include that proteases are often required to expose HS chains for heparanase degredation. Gel filtration analysis is difficult to adopt for high throughput. The use of radiolabeled material involves the disposition of material in accordance with safety standards.
Satoh et al. (32) report methods to covalently link oligosaccharides to methyl vinyl ether-maleic anhydride copolymer (MMAC) coated microtiter plates. These methods are cumbersome because they require several chemical reactions to link oligosaccharides onto MMAC coated plates. Some of the organic reagents and solvents may destroy microtiter plates. Furthermore, the overall yield could be very low after several steps of chemical reactions.
Freeman and Parish report a rapid method for measuring heparanase activity (27). Chicken histidine-rich glycoprotein (cHRG) was coupled to Sepharose and used for isolating undigested HS. Although the method separates heparanase-cleaved products from the HS substrate, the method requires large amounts of cHRG-Sepharose to quantitatively deplete undigested HS from the reaction mixtures. Also, the handling of Sepharose beads in connection with column chromatography and centrifugation methods does not lend itself to high throughput screening.
Ben-Artzi et al. (33) report a high throughput assay for measuring heparanase activity. Using HS or certain types of heparin species as a substrate, the heparanase reaction is carried out in 96 well microtiter plates and stopped by the addition of tetrazolium blue. The tetrazolium blue reacts with the reactive ends of sugars exposed by heparanase cleavage. The number of cleavages of the substrate is measured calorimetrically. The assay requires the use of large amounts of HS as substrate (50 μg HS per 100 μl) and at least a 2 hour heparanase incubation period (2–24 hrs). These requirements of the assay, though not procedurally optimal, provide conditions that favor cleavage of sugars of the HS substrate. The measurement of greater numbers of reactive sugars is necessary to compensate to some extent for the low signal to background ratio. A further drawback of the assay stems from the use of tetrazolium salt which is reactive with many types of groups, and can thus cause inaccurate results. Purity of assay reagents and test compounds is therefore a concern.
Heparanase catalytic activity could also be inhibited indirectly by an agent that prevents or alters heparanase substrate binding to FGF. Because such agents can potentially block FGF mediated cell signaling events, they are potential candidates as therapeutic agents for cancer and diseases caused by abnormal neovascularization (31).